Breast cancer is the second leading cause of cancer-related death in women in the US. Although it is still hotly debated, emerging evidence suggests that a small subset of breast cancer stem cells (BCSCs) might be responsible for tumor growth, metastases and resistance to current therapies and they share many characteristics with normal mammary stem cells (MaSCs). Thus, the study of MaSCs and BCSCs will help to elucidate the origin and the development of breast cancer, and might overcome its resistance to current therapies. During last 10 years of this grant, we have identified a unique phosphorylation-specific prolyl isomerase Pin1 as a key regulator of cell signaling in breast and other cancers. Pin1 is over expressed and correlates with poor patient outcome in many cancers including breast cancer. Furthermore, Pin1 has been shown to have profound impact on cell proliferation and transformation by acting on over 25 oncogenes and tumor suppressors. Moreover, Pin1 is pivotal for breast cancer development in vitro and in vivo. However, although several Pin1 substrates have well-known regulatory roles in MaSCs and BCSCs, little is known about the role of Pin1 in these stem cells. Our preliminary results showed that inhibition and over expression of Pin1 resulted in decreased and increased subpopulations and self-renewal activity of mouse MaSCs and/or tumorigenicity of human BCSCs, respectively, and our bioinformatics and genome-wide expression profiling analyses identified novel Pin1 regulators and potentially new Pin1 downstream targets important in these stem cells. These results led us to hypothesize that Pin1 plays an important role in regulating the self-renewal and differentiation and/or tumorigenicity of MaSCs and BCSCs. To test this novel hypothesis, we will first determine the role of Pin1 in the self-renewal and differentiation of normal MaSCs in vitro and in vivo using our germ line Pin1 KO, conditional Pin1 KO, and MMTV-Pin1 transgenic mice. Next, we will determine the role of Pin1 in the self-renewal and tumorigenicity of BCSCs by manipulating Pin1 function in established cell lines and primary cultures of human breast cancer, as well as crossing Pin1 KO or MMTV-Pin1 mice with MMTV-Neu or -Wnt1 mice. Finally, we will elucidate the mechanisms by which Pin1 regulates MaSCs and BCSCs by investigating how Pin1 is regulated in stem cells and how Pin1 executes its role in stem cells by determining some known Pin1 substrates and identifying new Pin1 downstream targets. Thus, these proposed experiments will use comprehensive approaches including cell and animal models and fresh human cancer tissues to elucidate for the first time the role and underlying mechanisms of Pin1 in regulating the self-renewal, differentiation and/or tumorigenicity of MaSCs and BCSCs. Successful completion of our studies would not only provide novel insight into breast cancer development, but also might have novel therapeutic implications because Pin1 inhibitors, which are being developed actively by pharmaceutical companies and research laboratories including ours, might be used to develop new therapies to achieve long-term remission of breast cancer and other cancers. PUBLIC HEALTH RELEVANCE: During last 10 years of this grant, we have identified a unique enzyme called Pin1 as a key regulator of cell signaling in breast and other cancers. The current proposal will investigate the role of Pin1 in controlling the function of breast cancer stem cells,a small subset of breast cancer cells that might be responsible for tumor growth, metastases and resistance to therapies. These studies would lead to a better understanding of the basic mechanisms of breast cancer development and might help develop new and more effective therapies to overcome cancer resistance to current therapies.